Genes for the subunits of human farnesyl-protein transferase

ABSTRACT

The present invention relates to an assay useful in determining the farnesyl-protein transferase inhibitory activity of pharmaceutical agents. The assay employs purified human farnesyl-protein transferase which is prepared by gene expression in Escherichia coli.

RELATED APPLICATIONS

The present patent application is a 371 filing of PCT International U.S.Ser. No. 93/10442, filed Oct. 29, 1993, published as WO94/10184 May 11,1994, which is a continuation application of application Ser. No.07/968,782, filed Oct. 30, 1992 now abandoned.

BACKGROUND OF THE INVENTION

The ras gene is found activated in many human cancers, includingcolorectal carcinoma, exocrine pancreatic carcinoma, and myeloidleukemias. Biological and biochemical studies of Ras action indicatethat Ras functions like a G-regulatory protein, since ras must belocalized in the plasma membrane and must bind with GTP in order totransform cells (Gibbs, J. et al., Microbiol. Rev., 53, 171-286 (1989)).Forms of ras in cancer cells have mutations that distinguish the encodedprotein from Ras in normal cells.

At least 3 post-translational modifications are involved with Rasmembrane localization, and all 3-modifications occur at the C-terminusof Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or"Cys-Aaa1-Aaa2-Xaa" box (Aaa is an aliphatic amino acid and Xaa is anyamino acid) (Willumsen et al., Nature, 310, 583-586(1984)). Otherproteins having this motif include the Ras-related GTP-binding proteinssuch as Rho, fungal mating factors, the nuclear lamins, and the gammasubunit of transducin.

Farnesyl-protein transferase (FPTase) catalyzes the addition of theisoprenoid farnesyl, from farnesyl diphosphate, to a cysteine residue ofsuch protein substrates having the CAAX terminus (Reiss, Y., Goldstein,J. L., Seabra, M. C., Casey, P. J. and Brown, M. S. (1990) Cell 62,81-88; Schaber, M. D., O'Hara, M. B., Garsky, V. M., Moser, S. D.,Bergstrom, J. D., Moores, S. L., Marshall. M. S., Friedman, P. A.,Dixon, R. A. F. and Gibbs, J. B. (1990) J. Biol. Chem. 265, 14701-14704;Manne, V., Roberts, D., Tobin, A., O'Rourke, E., De Virgilio, M.,Meyers, C., Ahmed, N., Kurz, B., Resh, M., Kung, H.-F. and Barbacid, M.(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7541-7545). Farnesylation ofRas facilitates its membrane binding which is essential for efficientcell transformation by oncogenic forms of Ras (Willumsen, B. M., Norris,K., Papageorge, A. G., Hubbert, N. L. and Lowy, D. R. (1984) EMBO J. 3,2581-2584). Thus, inhibitors of FPTase may be antitumor agents. CAAXtetrapeptides are substrates for FPTase with kinetic properties similarto polypeptide substrates indicating that the critical determinantsrequired for enzyme recognition are contained within the CAAX box (Reisset al., Cell, supra; Schaber et al., J. Biol. Chem., supra; Pompliano,D. L., Rands, E., Schaber, M. d., Mosser, S. D., Anthony, N. J. andGibbs, J. B. (1992) Biochem. 31, 3800-3807; Goldstein, J. L. Brown, M.S., Stradley, S. J., Reiss, Y. and Gierasch, L. M. (1991) J. Biol. Chem.266, 15575-15578).

Mammalian FPTase is an αβ heterodimeric protein. Complementary DNAsencoding part or all of the α subunit of FPTase from bovine and ratbrain have been isolated (Kohl, N. E. et al., (1991) J. Biol. Chem. 266,18884-18888; Chen et al. (1991) Cell, 66, 327-334; Chen et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88, 11368-11372). The proteins that theyencode share >95% amino acid sequence identity with one another and 30%identity with the RAM2-encoded subunit of Saccharomyces cerevisiaeFPTase (He, B. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88,11373-11377; Kohl et al., supra). A cDNA encoding the β subunit of ratbrain FPTase has been isolated and the protein it encodes shares 37%amino acid sequence identity with the DPR1/RAM1 (RAM1) encoded subunitof S. cerevisiae FPTase (Goodman, L. E. et al. (1988) Yeast, 4, 271-281;Chen et al. (1991) Cell, 66, 327-334) However these cDNAs have not beenused for expression of the complete FPTase that is readily isolatable inlarge quantities.

Previously, amounts of FPTase useful in assessing the inhibitoryactivity of pharmaceutical agents have been isolated from animal tissuesuch as rat or bovine brain. However, relatively low amounts of FPTaseare present in rat and bovine brain and purification of the enzymeinvolves a number of separation steps.

Human FPTase, the ideal enzyme to employ in an assay directed atdiscovering a human anticancer agent, has not been employed previouslybecause of the limited amounts of human FPTase available from human cellculture lines and human tissue and the hazards of working with humancells and tissue.

SUMMARY OF THE INVENTION

The instant invention provides a transformed bacterial cell line capableof expressing a modified mammalian farnesyl-protein transferase (FPTase)especially human farnesyl-protein transferase.

The instant invention also provides a method of preparing and purifyinga mammalian FPTase in experimentally useful quantities which comprisesexpressing the modified mammalian farnesyl-protein transferase andpurifying the modified FPTase on an antibody column.

The instant invention also provides an assay useful for thedetermination of FPTase inhibitory activity of pharmaceutical agentswhich comprises contacting the pharmaceutical agent to be assessed withsubstantially pure mammalian FPTase.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A,1B,1C: DNA sequence of the bovine cDNA that encodes the βsubunit of bovine brain FPTase enzyme. The numbering of the DNA sequenceis on the left of each line of DNA sequence. The numbering of the aminoacid sequence is at the right of each line of amino acid sequence.

FIGS. 2A,2B,2C: DNA sequence of the human cDNA that encodes the αsubunit of human FPTase enzyme. The numbering of the DNA sequence is onthe left of each line of DNA sequence. The numbering of the amino acidsequence is at the right of each line of amino acid sequence.

FIGS. 3A,3B,3C: DNA sequence of the human cDNA that encodes the βsubunit of human FPTase enzyme. The numbering of the DNA sequence is onthe left of each line of DNA sequence. The numbering of the amino acidsequence is at the right of each line of amino acid sequence.

FIG. 4: The plasmid, pRD426, which contains a cDNA sequence encoding theβ subunit of bovine FPTase. The restriction sites for the enzymes EcoRIand SspI are indicated. The distances between the restriciton sites areindicated in kilobases (kB). The part of the plasmid encoding the βsubunit of bovine FPTase is also indicated by an arc.

FIGS. 5A,5B,5C,5D,5E: DNA sequence of the insert used to generate thepRD516 plasmid which incorporates the human cDNAs that encode the α andβ subunits of human FPTase enzyme. The numbering of the DNA sequence ison the left of each line of DNA sequence. The numbering of the aminoacid sequence is at the right of each line of amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

A bacterial cell is transformed with a plasmid containing cDNAs encodingα and β subunits of a mammalian FPTase operationally linked by codons inone subunit which contain the ribosomal binding site necessary fortranslation of the other subunit by the bacterial cell.

The bacterial cell may be any cell capable of expressing thetranslationally coupled FPTase subunits. Such a cell line may beselected from strains of Escherichia coli, such as DH5, HB101, BL21(DE3)and the like.

The cDNAs encoding the α and β subunits of a mammalian FPTase may beselected from subunits such as the a and β subunits of human FPTase,bovine FPTase or rat FPTase and the like. The cDNA encoding an α subunitof one of the mammalian FPTases, for example human FPTase, may becombined with the cDNA encoding the β subunit of a different mammalianFPTase, for instance bovine FPTase. The preferred combination of cDNAsis the cDNAs encoding for the α and β subunits of human FPTase.

The plasmid vector into which the cDNAs encoding the α and β subunits ofa mammalian FPTase are cloned may be any vector compatable withtransformation into the selected bacterial cell line. Such vectorsinclude, but are not limited to, derivatives of ColE1 (such as pBR322,pUC8, pUC9, pUC18, pUC19, and the like) (Yanish-Perron et al, 1985,Gene, 33:103-119) or P15a (such as pACYC177, pACYC184, and the like) (A.C. Y. C. Chang and S. N. Cohen, 1978, J. Bacteriology, 134:1141-1156).To operationally link the cDNAs encoding the α and β subunits of amammalian FPTase to the plasmid, a promoter sequence is necessary. Suchpromoters include, but are not limited to, the tac promoter (E. Ammon etal., 1983, Gene, 25:167-178), lac promoter (U. Siebenlist etal., 1980,Cell, 20: 269-281) and trp promoter (G. N. Bennet et al., 1978, J. Mol.Biol., 121:113-137). Such promoters use E. coli RNA polymerase totranscribe DNA into mRNA. In one embodiment of the instant invention thepromoter is the tac promoter found in the plasmid pBTac1 (commerciallyavailable from Boehringer Mannheim Biochemicals). Other promoter/RNApolymerase systems useful in carrying out the instant invention includeDNA bacteriophage promoters and their cognate RNA polymerase. Examplesof such systems include the bacteriophage T7 promoter and T7 RNApolymerase (F. W. Studier and B. A. Moffat, 1986, J. Mol. Biol., 189:113-130). It is expected that the bacteriophage T7 promoter and RNApolymerase would give higher levels of protein expression than the tacpromoter.

To ensure translation of the transcribed cDNA sequences, a ribosomalbinding site must be operationally linked to the FPTase α and β subunitcoding sequences. An example of a ribosomal binding site for the βsubunit is GGAGG while a ribosomal binding for the α subunit is GGAGencoded in the Glu-Glu-Phe epitope tag which can be placed at theC-terminal end of the β subunit coding sequence.

The transformed cell line is grown and harvested and the mammalianFPTase expressed by the cells is isolated and purified. Isolation andpurification of the mammalian FPTase may be accomplished by any of thetechniques well known to persons skilled in the art. Preferably anepitope tag is incorporated in the FPTase when it is expressed and thecell lysate is exposed to a column containing an antibody which binds tothe tagging amino acid sequence. Most preferably the FPTase is taggedwith a C-terminal EEF epitope and the column utilized is a columncontaining monoclonal antibody YL1/2 (described in J. V. Kilmartin etal., J. Cell Biology, 93: 576-582(1982)).

The purified, recombinantly expressed, mammalian FPTase is employed inan assay to determine the inhibitory activity against FPTase mediatedfarnesylation of Ras by various pharmaceutical compounds. Ras protein isexposed to a labelled farnesyl diphosphate, such as ³ H!farnesyldiphosphate and the like, in the presence of the pharmaceutical compoundwhose activity is to be determined and in a suitable incubation medium,such as a mixture of 50 mM HEPES (pH 7.5), 5 mM MgCl₂ and 1-2 mMdithiothreitol, and the like. The purified mammalian FPTase is thenadded to the assay mixture and the assay mixture is incubated for aperiod of time such as 10-30 minutes at 30° C. The assay mixture is thenquenched and the Ras protein is separated from free FPP by techniqueswell known in the art. (see for example M. D. Schaber et al. (1990) J.Biol. Chem. 265, 14701-14704 and S. L. Moores et al. (1991), J. Biol.Chem., 266: 14603-14610) The isolated Ras protein is then analyzed by ascintillation counting technique to provide a relative value for theamount of Ras protein that had been farnesylated. This value may becompared to the relative value of the amount of Ras farnesylated in theabsence of the pharmaceutical agent and to the relative value of theamount of Ras farnesylated in the presence of other pharmaceuticalagents.

The invention is further defined by reference to the following examples,which are illustrative and not limiting. All temperatures are in degreesCelsius.

EXAMPLE 1

Labelled Probe from cDNA Encoding for the α Subunit of Bovine FPTase

A probe is used comprising an approximately 1000 bp DNA fragment thatspans the open reading frame of the α subunit of bovine FPTase describedby Kohl et al. J. Biol. Chem. 266, 18884-18888 (1991). This fragment wasmade by polymerase chain reaction (PCR) and has as its ends: (5' end:seq ID 1) ##STR1##

EXAMPLE 2

cDNA Encoding for the β Subunit of Bovine FPTase

Two overlapping, complimentary oligonucleotides5'-ATCCAGGCCACCACCCACTTCCTGCAGAAGCCT 3' (seq ID 2) and5'-CTCCTCAAAGCCAGGCACAGGCTTCTGCAGGAA-3' (seq ID 3) were made based oncodon preferrence usage (Lathe, R. (1985) J. Mol. Biol. 183, 1-12) forthe codons of the peptide IQATTHFLQKPVPGFEE (seq ID 4) from the βsubunit of rat brain FPTase (Reiss, Y., Seabra, M. C., Armstrong, S. A.,Slaughter, C. A., Goldstein, J. L. and Brown, M. S. J. Biol. Chem. 266,10672-10677 (1991)). These two oligonucleotides were annealed and filledin with the Klenow fragment of DNA polymerase and all four ³²P!-labelled deoxynucleotide triphosphates for use as a probe. The doublestranded fragment was then heated to separate the strands. A bovinebrain oligo(dT) primed cDNA library in λgt10 was screened using theplaque hybridization method and employing this probe. Filters fromplaque lifts of the bovine brain cDNA library were prehybridized andhybridized in 5× SSC, 10× Denhardt's solution (1× Denhardt's solution is20 mg/ml bovine serum albumin, 20 mg/ml polyvinylpyrollidone, 20 mg/mlFicoll), 0.1% (wt./vol.) SDS at 50° C. overnight. The filters werewashed in 5× SSC at 50° C. and autoradiographed. Positive plaques wereselected, grown up and the DNA analyzed by restriction and sequenceanalysis.

EXAMPLE 3

Labelled Probe from cDNA Encoding for the β Subunit of Bovine FPTase

A probe comprising an approximately 1660 bp EcoRI-SspI DNA fragment frompRD426 encoding the bovine β subunit cDNA obtained as described inExample 2 was prepared by restriction digestion. The restriction sitesare shown in FIG. 4. The EcoRI site, upstream of the start of the cDNA,comes from the linker used in the cDNA cloning, while the SspI site isapproximately 300 bp downstream of the stop codon for the β subunitcoding sequence in the nontranslated region of the cDNA. The fragmenthas the following structure: ##STR2##

EXAMPLE 4

cDNA Encoding for the α Subunit of Human FPTase

A human placental cDNA library in λgt11 (Clonetech) was probed using thelabelled DNA fragment from the bovine FPTase cDNA described in Example 1above. Filters from the plaque lifts of the human placental cDNA librarywere prehybridized and hybridized as described in Example 2 except at65° C. The filters were then washed as described in Example 2 at 65° C.Positive clones from the screens were utilized as described in Example 7below.

EXAMPLE 5

cDNA Encoding for the β Subunit of Human FPTase

A human placental cDNA library in λgt11 (Clonetech) was probed using thelabelled DNA fragment from the bovine FPTase cDNA described in Example 3above. Filters from the plaque lifts of the human placental cDNA librarywere prehybridized and hybridized as described in Example 2 except at65° C. The filters were then washed as described in Example 2 at 65° C.Positive clones from the screens were utilized as described in Example 7below.

EXAMPLE 6

DNA Sequencing of cDNAs

The cDNAs obtained as described in Examples 2, 4 and 5 were sequencedusing the Sequenase II dideoxy sequencing kit (U.S. Biochemical Corp.)as described by the manufacturer using plasmid DNAs as templates. DNAand protein sequence analysis was performed using the Genetics ComputerGroup software package and the multiple sequence alignment programCLUSTAL (Devereux, J. et al. (1984) Nucl. Acids Res. 12, 387-395 andHiggins, D. G. and Sharp, P. M. (1988) Gene, 73, 237-244.) DNAsubcloning, PCR and other DNA manipulations were performed as describedin Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Saiki, R. K., Gelfand, D. H., Stoffel, S. Scharf, S.J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A. (1988)Science 239, 487-491. The DNA sequences of the FPTase cDNAs weresubmitted to GenBank and were given the accession numbers L00633 (bovineβ subunit) (cDNA: seq ID 5; amino acid: seq ID 6), L00634 (human αsubunit) (cDNA: seq ID 7; amino acid: seq ID 8) and L00635 (human βsubunit) (cDNA: seq ID 9;amino acid: seq ID 10). FIGS. 1-3 show therespective sequences.

EXAMPLE 7

Construction of Plasmid for Expression of hFPTase in E. coli

The coding region of the α subunit of human FPTase was modified by PCR(Saiki et al.) such that EcoRI sites were placed immediately upstreamand downstream of the α subunit coding sequence and cloned into pUC18 tocreate pRD452. The sequence of the insert of pRD452 is as follows:##STR3## pRD452 was cut with SacI, which only cuts in the polylinkerregion of the plasmid, the ends were blunted with T4 DNA polymerase anddNTPs and then recircularized. This plasmid was partially digested withEcoRI, the ends filled in with the Klenow fragment of E. coli DNApolymerase and dNTPs and recircularized. A plasmid from this step, inwhich the EcoRI site down stream of the coding sequence of the α subunitof human FPTase was filled in, was identified and is named pRD494. Thesequence of the insert in pRD494 is the following: (3' end: seq ID 11)##STR4## The SacI restriction endonuclease site in this plasmid wascompletely lost. Synthetic oligonucleotides encoding an approximately 70bp EcoRI-BanII fragment at the 5'-end of the α subunit coding sequencewere made that contained primarily A or T at the third position of eachcodon, and inserted into pRD494 that had been cut with EcoRI and BanIIto create pRD493. The sequence of the α subunit coding sequence and thesurrounding restriction sites in pRD493 are as follows: (5' end: seq ID12; 3' end: seq ID 11) ##STR5## Using PCR, the sequence upstream of thestart codon of the α subunit in pRD493 was changed to include a ScaIsite immediately upstream of the start codon. The sequence of the αsubunit coding sequence and the surrounding restriction sites in pRD515are as follows: (5' end: seq ID 13; 3' end: seq ID 11) ##STR6## The 5'end of the cDNA encoding the β subunit of hFPTase was changed using PCRsuch that an EcoRI site was placed immediately upstream of the codingsequence. The 3' end of the cDNA for the β subunit was changed using PCRsuch that the codons for Glutamic acid-Glutamic acid-Phenylalanine-stopwere placed after the last codon for the β subunit. Additionally, a PacIsite was placed spanning the stop codon and an EcoRI site was placeddownstream of the PacI restriction site. This EcoRI fragment was clonedinto the EcoRI site of pBCKS+ (Stratagene) to make pRD463. The insertand the adjoining sequences in pRD463 are as follows: (5' end: seq ID14; 3' end: seq ID 15) ##STR7## The 5' end of the cDNA encoding the βsubunit of hFPTase was modified by PCR such that an EcoRI site wasplaced immediately adjacent to the start codon and a 0.7 kb EcoRI-KpnIfragment containing codons for aa₁ -aa₂₄₂ of the β subunit was clonedinto EcoRI+KpnI cut pUC18 creating pRD464. The ends of the insert inpRD464 are as follows: ##STR8## The 0.7 kb EcoRI-KpnI fragment of pRD464containing the 5' end of the β subunit gene and the 0.6 kb KpnI-HindIIIfragment from pRD463 containing the 3' end of the β subunit gene werecloned into the expression vector pBTac1 (Boehringer-Mannheim) that hadbeen cut with EcoRI and HindIII creating pRD466. The ends of the insertin pRD466 are as follows: (3' end: seq ID 16) ##STR9##

The plasmid pRD466 was partially cleaved with EcoRI and the ends filledin with the Klenow fragment of E. coli DNA polymerase and dNTPs, theends religated and transformed into E. coli. A plasmid from thistransformation was identified in which the EcoRI site after the βsubunit stop codon was filled in, while the EcoRI site upstream of the βsubunit coding sequence was intact. The insert in this plasmid,designated pRD478, has the following sequence: (3' end: seq ID 17)##STR10## The 5' end of the β subunit coding sequence was modified usingPCR and a ribosomal binding site (Shine, J. and Dalgarno, L. (1974)Proc. Natl. Acad. Sci. USA 71, 1342-1346) was placed between an EcoRIsite and the β subunit coding sequence. A 0.3 kb EcoRI-BamHI fragmentfrom this PCR was used to replace the EcoRI-BamHI fragment in pRD478,creating pRD486. The insert in pRD486 has the following sequence: (5'end: seq ID 18; 3' end: seq ID 17) ##STR11## The plasmid pRD486 wascleaved with PacI and the ends blunted with T4 DNA polymerase. This DNAwas ligated to a 1.1 kB ScaI-HincII fragment from pRD515 that containedthe coding sequence for the α subunit of hFPTase. In the resultingplasmid, pRD516, the coding sequences for the α and β subunits of humanFPTase are transcribed on a single mRNA driven by the tac promoter onthe plasmid. Translation of the α subunit is coupled (Schoner, B. E.,Belagaje, R. M. and Schoner, R. G. (1990) Methods in Enz. 185, 94-103)to the translation of the β subunit since the ribosomal binding site(RBS) for the α subunit is the sequence GGAG (Shine, J. and Dalgarno,L., supra) that is within the Glu-Glu-Phe coding sequence appended ontothe β subunit coding sequence. The sequence of the DNA fragmentcontaining the coding sequences for the α and β subunits of human FPTasein pRD516 is shown in FIG. 5. (cDNA: seq ID 19; amino acid: seq ID 20).

EXAMPLE 8

Expression and Purification of Human FPTase

To express human FPTase the plasmid pRD516 was transformed into E. coliDH5a and grown in LB+100 μg/ml ampicillin at 37° C. until the cultureswere in late log to early stationary phase of growth (This culture wasdeposited on Oct. 14, 1992, with the American Type Culture Collection at12301 Parklawn Drive, Rockville, Md. 20852 as ATCC-69091). The cellswere harvested and FPTase was purified as described below. Human FPTasewas isolated from E. coli cultures by resuspending a cell pellet in 50mM Tris-Cl pH7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mMphenylmethylsulfonyl flouride (PMSF), 2 μg/ml leupeptin, 2 μg/mlantipain, 10 μg/ml aprotinin (approx. 5 g cells/10 ml solution). Theresuspended cells were broken by sonication and the cell debris pelletedby centrifugation at 30,000×g at 4° C. for 30 min. The soluble fractionwas diluted with an equal volume of 0.15M NaCl, 6 mM NaPO₄, pH7.2 (PBS)and applied at a flow rate of approximately 0.5 ml/min to a 2 ml columnof the monoclonal antibody YL1/2(4 mg antibody/ml resin) coupled tocyanogen bromide activated Sepharose (Stammers et al.) After loading theprotein onto the column, the column was washed with 10-20 ml of 1/2×PBScontaining 2 mM DTT, 0.1% Tween-20, 1 mM PMSF, 2 μg/ml leupeptin, 2μg/ml antipain, 10 μg/ml aprotinin. The column was then washed with100-200 ml of 1/2×PBS containing 2 mM DTT. FPTase was eluted with 3×3 mlof 5 mM Asp-Phe dipeptide (Sigma), 100 mM Tris-Cl pH 7.5, 2 mM DTT. Thecolumn was regenerated by washing with PBS+2M NaCl and then storing inPBS+0.02% (wt./vol.) NaN₃. The FPTase was obtained in >20% purity and ina 0.1 to 1.0% yield based on the total starting soluble E. coli protein.In some cases the FPTase was further purified. This is not necessary fordrug screening. To further purify the FPTase he protein eluted from theantibody column was chromatographed by HPLC on a MonoQ HR10-10 column(Pharmacia) where buffer A was 50 mM Tris-Cl pH7.5, 5 mM MgCl₂, 5 mM DTTand buffer B was buffer A+1M NaCl. The column was run at 1 ml/min. andthe gradient was 0-20% B in 10 min., 20-40% B in 30 min., 40-100% B in20 min. FPTase eluted at approximately 30-35% B.

EXAMPLE 9

Assay of Purified FPTase Activity in the Absence of Inhibitor

Activity of the purified human FPTase described in Example 8 was assayedin the biosynthetically forward direction at 30° C. Reactions were neverallowed to proceed to more than 10% completion based on the limitingsubstrate. For calculations, the molecular mass of the transferase wasassumed to be 93 kD and that of Ras-CVLS to be 21 kD (D. L. Pompliano etal., Biochem. 31, 3800-3807 (1992) and S. L. Moores et al. (1991), J.Biol. Chem., 266: 14603-14610)). A typical reaction contained thefollowing: 50 mM HEPES pH7.5, 5 mM MgCl₂, 5 mM DTT, 10 mM ZnCl₂, 0.1%(wt./vol) polyethylene glycol (ave mol wt. 20000) with ³ H!-farnesyldiphosphate (1-3000 nM) and Ras-CVLS protein (20-10000 nM) as substrate.After thermally preequilibrating the asssay mixture in the absence ofthe enzyme, the reaction was initiated by adding the transferase.Aliquots (50-500 mL) were withdrawn at timed intervals and diluted into10% HCl in ethanol (1-2 mL). The quenched reaction aliquots were allowedto stand at room temperature for 15 minutes (to hydrolyze unreactedfarnesyl diphosphate). After adding 100% ethanol (2 ml), the aliquotworkups were vacuum filtered through Whatman GF/C filters using aBrandel cell harvester. Filters were washed four times with 2 mL 100%ethanol, mixed with scintillation fluid (10 mL) and then counted in aBeckman LS3801 scintillation counter. Kinetic data was obtained for thepurified human FPTase obtained as described in Example 8 and bovinebrain FPTase, obtained as described in S. L. Moores et al., J. Biol.Chem., 266, 14603 (1991) and is listed in Table 1.

                  TABLE 1                                                         ______________________________________                                        FPTase source                                                                            FPP K.sub.m (nM)                                                                        Ras-CVLS K.sub.m (nM)                                                                      k.sub.cat (s.sup.-1)                        ______________________________________                                        human pFPTase-αβ                                                              9.8 ± 1.7                                                                            392 ± 20  0.010 ± 0.001                            bovine brain                                                                             9.3 ± 1.0                                                                            620 ± 80  0.013 ± 0.001                            pFPTase                                                                       ______________________________________                                    

EXAMPLE 10

Assay of Compounds for FPTase Inhibitory Activity

³ H!Farnesyl diphosphate (FPP) (20 Ci/mmol) was purchased from NewEngland Nuclear. In general, inhibitory activity assays were carried outat 30° C. A typical assay reaction contained (in a final volume of 50μL): ³ H!farnesyl diphosphate, Ras protein (3.5 μM), 50 mM HEPES, pH7.5, 5 mM MgCl₂, 1 mM dithiothreitol, and the compound to be assayed.After thermally preequilibrating the assay mixture, the reaction wasinitiated by the addition of the purified mammalian FPTase and thereaction was stopped at timed intervals by the addition of 1M HCl inethanol (1 mL). The quenched reactions were allowed to stand for 15minutes. After adding 2 mL of 100% ethanol, the mixtures were vacuumfiltered through Whatman GF/C filters. Filters were washed four timeswith 2 mL aliquots of 100% ethanol, mixed with scintillation fluid (10mL) and then counted in a Beckmann LS3801 scintillation counter.Inhibitory activity values for known compounds in the assay employingthe once purified human FPTase and an assay employing bovine brainFPTase are compared in Table 2 below:

                  TABLE 2                                                         ______________________________________                                        Inhibitor     IC.sub.50 value (nM)                                            FTase         Bovine brain FTase                                                                         Human                                              ______________________________________                                        tetrapeptide  100          100                                                CVIM *                                                                        tetrapeptide  10           17                                                 CIFM                                                                          tetrapeptide  20,000       6,200                                              CVIL *                                                                        unlabelled                                                                    FPP *         200          250                                                unlabelled                                                                    GGPP          200          240                                                Compound A    20           18                                                 ______________________________________                                         * = alternative substrates                                                    CVIM = cysteinylvalinyl-isolleucinyl-methionine                               CIFM = cysteinylisolleucinyl-phenylalaninyl-methionine                        CVIL = cysteinylvalinyl-isolleucinyl-leucine                                  GGPP = geranylgeranyl-diphosphate                                             Compound A =                                                                  (R,S) 1hydroxy-(E,E)-3,7,11-trimethyl-2,6,10-dodeca-trienyl!phosphonic        acid (Pomphiano, D. L. et al., Biochem. 31, 3800-3807 (1992)).           

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 22                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AAGCTTAACCATGGACGACGGG22                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATCCAGGCCACCACCCACTTCCTGCAGAAGCCT33                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTCCTCAAAGCCAGGCACAGGCTTCTGCAGGAA33                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       IleGlnAlaThrThrHisPheLeuGlnLysProValProGlyPheGlu                              151015                                                                        Glu                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1314 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGGCTTCTCCGAGTTCCTTCACCTACTGTTGCCCTCCATCTTCCTCCCCTATCTGGTCA60                GAACCGCTGTACAGTCTGAGGCCAGAGCACGCGCGGGAGCGGTTGCAGGACGACTCGGTG120               GAAACAGTCACGTCCATAGAACAGGCAAAAGTAGAAGAAAAGATCCAAGAGGTCTTCAGT180               TCTTACAAGTTCAACCACCTTGTACCAAGGCTTGTTTTGCAGAGAGAGAAGCACTTCCAT240               TATCTGAAAAGAGGCCTCCGACAGCTGACAGATGCCTACGAGTGTCTGGATGCCAGCCGC300               CCATGGCTCTGCTACTGGATCCTGCATAGCCTGGAACTCCTGGATGAGCCCATCCCCCAG360               ATGGTGGCCACAGACGTGTGTCAGTTCCTGGAGTTGTGTCAGAGCCCAGAAGGCGGCTTT420               GGAGGGGGCCCTGGCCAGTACCCACACCTTGCACCCACGTATGCAGCGGTCAACGCGCTG480               TGCATCATTGGCACCGAGGAGGCCTATGACGTCATTAACAGAGAGAAGCTTCTCCAGTAT540               TTGTACTCGCTGAAGCAACCCGATGGCTCTTTTCTCATGCACGATGGAGGTGAGGTGGAC600               GTGAGAAGTGCATACTGTGCTGCCTCGGTAGCTTCGTTGACCAACATCATCACCCCAGAC660               CTGTTTGAGGGCACTGCTGAATGGATCGCAAGGTGTCAGAATTGGGAAGGTGGGATTGGC720               GGGGTACCAGGAATGGAAGCCCATGGCGGCTACACGTTCTGTGGCCTGGCTGCGCTGGTC780               ATCCTCAAGAAGGAGCGCTCCTTGAACTTGAAGAGCTTACTACAATGGGTGACAAGCCGG840               CAGATGAGGTTTGAAGGTGGATTTCAGGGCCGCTGCAACAAGCTGGTAGACGGCTGCTAC900               TCCTTCTGGCAGGCGGGTCTCCTGCCCCTGCTTCACCGCGCGCTGCACGCCCAAGGTGAC960               CCTGCCCTCAGCATGAGTCGCTGGATGTTTCACCAGCAGGCCCTGCAGGAGTACATCCTG1020              ATGTGCTGCCAGTGCCCCACCGGGGGGCTTCTGGACAAACCTGGCAAGTCCCGGGACTTC1080              TACCACACCTGCTACTGCCTGAGTGGCCTGTCCATAGCCCAGCACTTCGGCAGCGGAGCC1140              ATGTTGCACGATGTGGTCTTGGGTGTACCTGAAAACGCCCTGCAGCCCACTCACCCTGTG1200              TACAATATTGGACCAGACAAAGTGATCCAGGCTACCATGCACTTTCTGCAGAAGCCAGTT1260              CCAGGCTTTGAGGAGCATGAGGATGAGGCATCAGCAGAGCCTGCCACTGACTAG1314                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 437 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAlaSerProSerSerPheThrTyrCysCysProProSerSerSer                              151015                                                                        ProIleTrpSerGluProLeuTyrSerLeuArgProGluHisAlaArg                              202530                                                                        GluArgLeuGlnAspAspSerValGluThrValThrSerIleGluGln                              354045                                                                        AlaLysValGluGluLysIleGlnGluValPheSerSerTyrLysPhe                              505560                                                                        AsnHisLeuValProArgLeuValLeuGlnArgGluLysHisPheHis                              65707580                                                                      TyrLeuLysArgGlyLeuArgGlnLeuThrAspAlaTyrGluCysLeu                              859095                                                                        AspAlaSerArgProTrpLeuCysTyrTrpIleLeuHisSerLeuGlu                              100105110                                                                     LeuLeuAspGluProIleProGlnMetValAlaThrAspValCysGln                              115120125                                                                     PheLeuGluLeuCysGlnSerProGluGlyGlyPheGlyGlyGlyPro                              130135140                                                                     GlyGlnTyrProHisLeuAlaProThrTyrAlaAlaValAsnAlaLeu                              145150155160                                                                  CysIleIleGlyThrGluGluAlaTyrAspValIleAsnArgGluLys                              165170175                                                                     LeuLeuGlnTyrLeuTyrSerLeuLysGlnProAspGlySerPheLeu                              180185190                                                                     MetHisAspGlyGlyGluValAspValArgSerAlaTyrCysAlaAla                              195200205                                                                     SerValAlaSerLeuThrAsnIleIleThrProAspLeuPheGluGly                              210215220                                                                     ThrAlaGluTrpIleAlaArgCysGlnAsnTrpGluGlyGlyIleGly                              225230235240                                                                  GlyValProGlyMetGluAlaHisGlyGlyTyrThrPheCysGlyLeu                              245250255                                                                     ArgAlaLeuValIleLeuLysLysGluArgSerLeuAsnLeuLysSer                              260265270                                                                     LeuLeuGlnTrpValThrSerArgGlnMetArgPheGluGlyGlyPhe                              275280285                                                                     GlnGlyArgCysAsnLysLeuValAspGlyCysTyrSerPheTrpGln                              290295300                                                                     AlaGlyLeuLeuProLeuLeuHisArgAlaLeuHisAlaGlnGlyAsp                              305310315320                                                                  ProAlaLeuSerMetSerArgTrpMetPheHisGlnGlnAlaLeuGln                              325330335                                                                     GluTyrIleLeuMetCysCysGlnCysProThrGlyGlyLeuLeuAsp                              340345350                                                                     LysProGlyLysSerArgAspPheTyrHisThrCysTyrCysLeuSer                              355360365                                                                     GlyLeuSerIleAlaGlnHisPheGlySerGlyAlaMetLeuHisAsp                              370375380                                                                     ValValLeuGlyValProGluAsnAlaLeuGlnProThrHisProVal                              385390395400                                                                  TyrAsnIleGlyProAspLysValIleGlnAlaThrMetHisPheLeu                              405410415                                                                     GlnLysProValProGlyPheGluGluHisGluAspGluAlaSerAla                              420425430                                                                     GluProAlaThrAsp                                                               435                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1140 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGGCGGCCACCGAGGGGGTCGGGGAGGCTGCGCAAGGGGGCGAGCCCGGGCAGCCGGCG60                CAACCCCCGCCCCAGCCGCACCCACCGCCGCCCCAGCAGCAGCACAAGGAAGAGATGGCG120               GCCGAGGCTGGGGAAGCCGTGGCGTCCCCCATGGACGACGGGTTTGTGAGCCTGGACTCG180               CCCTCCTATGTCCTGTACAGGGACAGAGCAGAATGGGCTGATATAGATCCGGTGCCGCAG240               AATGATGGCCCCAATCCCGTGGTCCAGATCATTTATAGTGACAAATTTAGAGATGTTTAT300               GATTACTTCCGAGCTGTCCTGCAGCGTGATGAAAGAAGTGAACGAGCTTTTAAGCTAACC360               CGGGATGCTATTGAGTTAAATGCAGCCAATTATACAGTGTGGCATTTCCGGAGAGTTCTT420               TTGAAGTCACTTCAGAAGGATCTACATGAGGAAATGAACTACATCACTGCAATAATTGAG480               GAGCAGCCCAAAAACTATCAAGTTTGGCATCATAGGCGAGTATTAGTGGAATGGCTAAGA540               GATCCATCTCAGGAGCTTGAATTTATTGCTGATATTCTTAATCAGGATGCAAAGAATTAT600               CATGCCTGGCAGCATCGACAATGGGTTATTCAGGAATTTAAACTTTGGGATAATGAGCTG660               CAGTATGTGGACCAACTTCTGAAAGAGGATGTGAGAAATAACTCTGTCTGGAACCAAAGA720               TACTTCGTTATTTCTAACACCACTGGCTACAATGATCGTGCTGTATTGGAGAGAGAAGTC780               CAATACACTCTGGAAATGATTAAACTAGTACCACATAATGAAAGTGCATGGAACTATTTG840               AAAGGGATTTTGCAGGATCGTGGTCTTTCCAAATATCCTAATCTGTTAAATCAATTACTT900               GATTTACAACCAAGTCATAGTTCCCCCTACCTAATTGCCTTTCTTGTGGATATCTATGAA960               GACATGCTAGAAAATCAGTGTGACAATAAGGAAGACATTCTTAATAAAGCATTAGAGTTA1020              TGTGAAATCCTAGCTAAAGAAAAGGACACTATAAGAAAGGAATATTGGAGATACATTGGA1080              AGATCCCTTCAAAGCAAACACAGCACAGAAAATGACTCACCAACAAATGTACAGCAATAA1140              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 379 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetAlaAlaThrGluGlyValGlyGluAlaAlaGlnGlyGlyGluPro                              151015                                                                        GlyGlnProAlaGlnProProProGlnProHisProProProProGln                              202530                                                                        GlnGlnHisLysGluGluMetAlaAlaGluAlaGlyGluAlaValAla                              354045                                                                        SerProMetAspAspGlyPheValSerLeuAspSerProSerTyrVal                              505560                                                                        LeuTyrArgAspArgAlaGluTrpAlaAspIleAspProValProGln                              65707580                                                                      AsnAspGlyProAsnProValValGlnIleIleTyrSerAspLysPhe                              859095                                                                        ArgAspValTyrAspTyrPheArgAlaValLeuGlnArgAspGluArg                              100105110                                                                     SerGluArgAlaPheLysLeuThrArgAspAlaIleGluLeuAsnAla                              115120125                                                                     AlaAsnTyrThrValTrpHisPheArgArgValLeuLeuLysSerLeu                              130135140                                                                     GlnLysAspLeuHisGluGluMetAsnTyrIleThrAlaIleIleGlu                              145150155160                                                                  GluGlnProLysAsnTyrGlnValTrpHisHisArgArgValLeuVal                              165170175                                                                     GluTrpLeuArgAspProSerGlnGluLeuGluPheIleAlaAspIle                              180185190                                                                     LeuAsnGlnAspAlaLysAsnTyrHisAlaTrpGlnHisArgGlnTrp                              195200205                                                                     ValIleGlnGluPheLysLeuTrpAspAsnGluLeuGlnTyrValAsp                              210215220                                                                     GlnLeuLeuLysGluAspValArgAsnAsnSerValTrpAsnGlnArg                              225230235240                                                                  TyrPheValIleSerAsnThrThrGlyTyrAsnAspArgAlaValLeu                              245250255                                                                     GluArgGluValGlnTyrThrLeuGluMetIleLysLeuValProHis                              260265270                                                                     AsnGluSerAlaTrpAsnTyrLeuLysGlyIleLeuGlnAspArgGly                              275280285                                                                     LeuSerLysTyrProAsnLeuLeuAsnGlnLeuLeuAspLeuGlnPro                              290295300                                                                     SerHisSerSerProTyrLeuIleAlaPheLeuValAspIleTyrGlu                              305310315320                                                                  AspMetLeuGluAsnGlnCysAspAsnLysGluAspIleLeuAsnLys                              325330335                                                                     AlaLeuGluLeuCysGluIleLeuAlaLysGluLysAspThrIleArg                              340345350                                                                     LysGluTyrTrpArgTyrIleGlyArgSerLeuGlnSerLysHisSer                              355360365                                                                     ThrGluAsnAspSerProThrAsnValGlnGln                                             370375                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1314 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGGCTTCTCCGAGTTCTTTCACCTACTATTGCCCTCCATCTTCCTCCCCCGTCTGGTCA60                GAGCCGCTGTACAGTCTGAGGCCCGAGCACGCGCGAGAGCGGTTGCAGGACGACTCGGTG120               GAAACAGTCACGTCCATAGAACAGGCAAAAGTAGAAGAAAAGATCCAAGAGGTCTTCAGT180               TCTTACAAGTTCAACCACCTTGTACCAAGGCTTGTTTTGCAGAGGGAGAAGCACTTCCAT240               TATCTGAAAAGAGGCCTTCGACAACTGACAGATGCCTATGAGTGTCTGGATGCCAGCCGC300               CCATGGCTCTGCTATTGGATCCTGCACAGCTTGGAACTGCTAGATGAACCCATCCCCCAG360               ATAGTGGCTACAGATGTGTGTCAGTTCCTGGAGCTGTGTCAGAGCCCAGAAGGTGGCTTT420               GGAGGAGGACCCGGTCAGTATCCACACCTTGCACCCACATATGCAGCAGTCAATGCATTG480               TGCATCATTGGCACCGAGGAGGCCTATGACATCATTAACAGAGAGAAGCTTCTTCAGTAT540               TTGTACTCCCTGAAGCAACCTGACGGCTCCTTTCTCATGCATGTCGGAGGTGAGGTGGAT600               GTGAGAAGCGCATACTGTGCTGCCTCCGTAGCCTCGCTGACCAACATCATCACTCCAGAC660               CTCTTTGAGGGCACTGCTGAATGGATAGCAAGGTGTCAGAACTGGGAAGGTGGCATTGGC720               GGGGTACCAGGGATGGAAGCCCATGGTGGCTATACCTTCTGTGGCCTGGCCGCGCTGGTA780               ATCCTCAAGAGGGAACGTTCCTTGAACTTGAAGAGCTTATTACAATGGGTGACAAGCCGG840               CAGATGCGATTTGAAGGAGGATTTCAGGGCCGCTGCAACAAGCTGGTGGATGGCTGCTAC900               TCCTTCTGGCAGGCGGGGCTCCTGCCCCTGCTCCACCGCGCACTGCACGCCCAAGGTGAC960               CCTGCCCTTAGCATGAGCCACTGGATGTTCCATCAGCAGGCCCTGCAGGAGTACATCCTG1020              ATGTGCTGCCAGTGCCCTGCGGGGGGGCTTCTGGATAAACCTGGCAAGTCGCGTGATTTC1080              TACCACACCTGCTACTGCCTGAGCGGCCTGTCCATAGCCCAGCACTTCGGCAGCGGAGCC1140              ATGTTGCATGATGTGGTCCTGGGTGTGCCCGAAAACGCTCTGCAGCCCACTCACCCAGTG1200              TACAACATTGGACCAGACAAGGTGATCCAGGCCACTACATACTTTCTACAGAAGCCAGTC1260              CCAGGTTTTGAGGAGCTTAAGGATGAGACATCGGCAGAGCCTGCAACCGACTAG1314                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 437 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetAlaSerProSerSerPheThrTyrTyrCysProProSerSerSer                              151015                                                                        ProValTrpSerGluProLeuTyrSerLeuArgProGluHisAlaArg                              202530                                                                        GluArgLeuGlnAspAspSerValGluThrValThrSerIleGluGln                              354045                                                                        AlaLysValGluGluLysIleGlnGluValPheSerSerTyrLysPhe                              505560                                                                        AsnHisLeuValProArgLeuValLeuGlnArgGluLysHisPheHis                              65707580                                                                      TyrLeuLysArgGlyLeuArgGlnLeuThrAspAlaTyrGluCysLeu                              859095                                                                        AspAlaSerArgProTrpLeuCysTyrTrpIleLeuHisSerLeuGlu                              100105110                                                                     LeuLeuAspGluProIleProGlnIleValAlaThrAspValCysGln                              115120125                                                                     PheLeuGluLeuCysGlnSerProGluGlyGlyPheGlyGlyGlyPro                              130135140                                                                     GlyGlnTyrProHisLeuAlaProThrTyrAlaAlaValAsnAlaLeu                              145150155160                                                                  CysIleIleGlyThrGluGluAlaTyrAspIleIleAsnArgGluLys                              165170175                                                                     LeuLeuGlnTyrLeuTyrSerLeuLysGlnProAspGlySerPheLeu                              180185190                                                                     MetHisValGlyGlyGluValAspValArgSerAlaTyrCysAlaAla                              195200205                                                                     SerValAlaSerLeuThrAsnIleIleThrProAspLeuPheGluGly                              210215220                                                                     ThrAlaGluTrpIleAlaArgCysGlnAsnTrpGluGlyGlyIleGly                              225230235240                                                                  GlyValProGlyMetGluAlaHisGlyGlyTyrThrPheCysGlyLeu                              245250255                                                                     AlaAlaLeuValIleLeuLysArgGluArgSerLeuAsnLeuLysSer                              260265270                                                                     LeuLeuGlnTrpValThrSerArgGlnMetArgPheGluGlyGlyPhe                              275280285                                                                     GlnGlyArgCysAsnLysLeuValAspGlyCysTyrSerPheTrpGln                              290295300                                                                     AlaGlyLeuLeuProLeuLeuHisArgAlaLeuHisAlaGlnGlyAsp                              305310315320                                                                  ProAlaLeuSerMetSerHisTrpMetPheHisGlnGlnAlaLeuGln                              325330335                                                                     GluTyrIleLeuMetCysCysGlnCysProAlaGlyGlyLeuLeuAsp                              340345350                                                                     LysProGlyLysSerArgAspPheTyrHisThrCysTyrCysLeuSer                              355360365                                                                     GlyLeuSerIleAlaGlnHisPheGlySerGlyAlaMetLeuHisAsp                              370375380                                                                     ValValLeuGlyValProGluAsnAlaLeuGlnProThrHisProVal                              385390395400                                                                  TyrAsnIleGlyProAspLysValIleGlnAlaThrThrTyrPheLeu                              405410415                                                                     GlnLysProValProGlyPheGluGluLeuLysAspGluThrSerAla                              420425430                                                                     GluProAlaThrAsp                                                               435                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TAAGAATTAATTC13                                                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GAATTCATGGCTGCTACTGAAGGTGTTGGTGAAGCTGCACAGGGTGGT48                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GAATTCATGACTATGGCTGCTACTGAAGGTGTTGGTGAAGCTGCACAGGGTGGT54                      (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GAATTCAACATG12                                                                (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GAGGAGTTTTAATTAAGAATTCGATATCAAGCTT34                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAGGAGTTTTAATTAAGAATTGATATCAAGCTT33                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GAGGAGTTTTAATTAAGAATTATTCGATATCAAGCTT37                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GAATTCTAAGGAGGAAAAAAAAATG25                                                   (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2546 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GAATTCTAAGGAGGAAAAAAAAATGGCTTCTCCGAGTTCTTTCACCTACTATTGCCCTCC60                ATCTTCCTCCCCCGTCTGGTCAGAGCCGCTGTACAGTCTGAGGCCCGAGCACGCGCGAGA120               GCGGTTGCAGGACGACTCGGTGGAAACAGTCACGTCCATAGAACAGGCAAAAGTAGAAGA180               AAAGATCCAAGAGGTCTTCAGTTCTTACAAGTTCAACCACCTTGTACCAAGGCTTGTTTT240               GCAGAGGGAGAAGCACTTCCATTATCTGAAAAGAGGCCTTCGACAACTGACAGATGCCTA300               TGAGTGTCTGGATCGGAGCCGCCCATGGCTCTGCTATTGGATCCTGCACAGCTTGGAACT360               GCTAGATGAACCCATCCCCCAGATAGTGGCTACAGATGTGTGTCAGTTCCTGGAGCTGTG420               TCAGAGCCCAGAAGGTGGCTTTGGAGGAGGACCCGGTCAGTATCCACACCTTGCACCCAC480               ATATGCAGCAGTCAATGCATTGTGCATCATTGGCACCGAGGAGGCCTATGACATCATTAA540               CAGAGAGAAGCTTCTTCAGTATTTGTACTCCCTGAAGCAACCTGACGGCTCCTTTCTCAT600               GCATGTCGGAGGTGAGGTGGATGTGAGAAGCGCATACTGTGCTGCCTCCGTAGCCTCGCT660               GACCAACATCATCACTCCAGACCTCTTTGAGGGCACTGCTGAATGGATAGCAAGGTGTCA720               GAACTGGGAAGGTGGCATTGGCGGGGTACCAGGGATGGAAGCCCATGGTGGCTATACCTT780               CTGTGGCCTGGCCGCGCTGGTAATCCTCAAGAGGGAACGTTCCTTGAACTTGAAGAGCTT840               ATTACAATGGGTGACAAGCCGGCAGATGCGATTTGAAGGAGGATTTCAGGGCCGCTGCAA900               CAAGCTGGTGGATGGCTGCTACTCCTTCTGGCAGGCGGGGCTCCTGCCCCTGCTCCACCG960               CGCACTGCACGCCCAAGGTGACCCTGCCCTTAGCATGAGCCACTGGATGTTCCATCAGCA1020              GGCCCTGCAGGAGTACATCCTGATGTGCTGCCAGTGCCCTGCGGGGGGGCTTCTGGATAA1080              ACCTGGCAAGTCGCGTGATTTCTACCACACCTGCTACTGCCTGAGCGGCCTGTCCATAGC1140              CCAGCACTTCGGCAGCGGAGCCATGTTGCATGATGTGGTCCTGGGTGTGCCCGAAAACGC1200              TCTGCAGCCCACTCACCCAGTGTACAACATTGGACCAGACAAGGTGATCCAGGCCACTAC1260              ATACTTTCTACAGAAGCCAGTCCCAGGTTTTGAGGAGCTTAAGGATGAGACATCGGCAGA1320              GCCTGCAACCGACGAGGAGTTTTAACTATGGCTGCTACTGAAGGTGTTGGTGAAGCTGCA1380              CAGGGTGGTGAGCCCGGGCAGCCGGCGCAACCCCCGCCCCAGCCGCACCCACCGCCGCCC1440              CAGCAGCAGCACAAGGAAGAGATGGCGGCCGAGGCTGGGGAAGCCGTGGCGTCCCCCATG1500              GACGACGGGTTTGTGAGCCTGGACTCGCCCTCCTATGTCCTGTACAGGGACAGAGCAGAA1560              TGGGCTGATATAGATCCGGTGCCGCAGAATGATGGCCCCAATCCCGTGGTCCAGATCATT1620              TATAGTGACAAATTTAGAGATGTTTATGATTACTTCCGAGCTGTCCTGCAGCGTGATGAA1680              AGAAGTGAACGAGCTTTTAAGCTAACCCGGGATGCTATTGAGTTAAATGCAGCCAATTAT1740              ACAGTGTGGCATTTCCGGAGAGTTCTTTTGAAGTCACTTCAGAAGGATCTACATGAGGAA1800              ATGAACTACATCACTGCAATAATTGAGGAGCAGCCCAAAAACTATCAAGTTTGGCATCAT1860              AGGCGAGTATTAGTGGAATGGCTAAGAGATCCATCTCAGGAGCTTGAATTTATTGCTGAT1920              ATTCTTAATCAGGATGCAAAGAATTATCATGCCTGGCAGCATCGACAATGGGTTATTCAG1980              GAATTTAAACTTTGGGATAATGAGCTGCAGTATGTGGACCAACTTCTGAAAGAGGATGTG2040              AGAAATAACTCTGTCTGGAACCAAAGATACTTCGTTATTTCTAACACCACTGGCTACAAT2100              GATCGTGCTGTATTGGAGAGAGAAGTCCAATACACTCTGGAAATGATTAAACTAGTACCA2160              CATAATGAAAGTGCATGGAACTATTTGAAAGGGATTTTGCAGGATCGTGGTCTTTCCAAA2220              TATCCTAATCTGTTAAATCAATTACTTGATTTACAACCAAGTCATAGTTCCCCCTACCTA2280              ATTGCCTTTCTTGTGGATATCTATGAAGACATGCTAGAAAATCAGTGTGACAATAAGGAA2340              GACATTCTTAATAAAGCATTAGAGTTATGTGAAATCCTAGCTAAAGAAAAGGACACTATA2400              AGAAAGGAATATTGGAGATACATTGGAAGATCCCTTCAAAGCAAACACAGCACAGAAAAT2460              GACTCACCAACAAATGTACAGCAATAAGAATTAATTCGGTACCCGGGGATCCTCTAGAGT2520              CTAAGAATTAATTCGATATCAAGCTT2546                                                (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 819 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetAlaSerProSerSerPheThrTyrTyrCysProProSerSerSer                              151015                                                                        ProValTrpSerGluProLeuTyrSerLeuArgProGluHisAlaArg                              202530                                                                        GluArgLeuGlnAspAspSerValGluThrValThrSerIleGluGln                              354045                                                                        AlaLysValGluGluLysIleGlnGluValPheSerSerTyrLysPhe                              505560                                                                        AsnHisLeuValProArgLeuValLeuGlnArgGluLysHisPheHis                              65707580                                                                      TyrLeuLysArgGlyLeuArgGlnLeuThrAspAlaTyrGluCysLeu                              859095                                                                        AspAlaSerArgProTrpLeuCysTyrTrpIleLeuHisSerLeuGlu                              100105110                                                                     LeuLeuAspGluProIleProGlnIleValAlaThrAspValCysGln                              115120125                                                                     PheLeuGluLeuCysGlnSerProGluGlyGlyPheGlyGlyGlyPro                              130135140                                                                     GlyGlnTyrProHisLeuAlaProThrTyrAlaAlaValAsnAlaLeu                              145150155160                                                                  CysIleIleGlyThrGluGluAlaTyrAspIleIleAsnArgGluLys                              165170175                                                                     LeuLeuGlnTyrLeuTyrSerLeuLysGlnProAspGlySerPheLeu                              180185190                                                                     MetHisValGlyGlyGluValAspValArgSerAlaTyrCysAlaAla                              195200205                                                                     SerValAlaSerLeuThrAsnIleIleThrProAspLeuPheGluGly                              210215220                                                                     ThrAlaGluTrpIleAlaArgCysGlnAsnTrpGluGlyGlyIleGly                              225230235240                                                                  GlyValProGlyMetGluAlaHisGlyGlyTyrThrPheCysGlyLeu                              245250255                                                                     AlaAlaLeuValIleLeuLysArgGluArgSerLeuAsnLeuLysSer                              260265270                                                                     LeuLeuGlnTrpValThrSerArgGlnMetArgPheGluGlyGlyPhe                              275280285                                                                     GlnGlyArgCysAsnLysLeuValAspGlyCysTyrSerPheTrpGln                              290295300                                                                     AlaGlyLeuLeuProLeuLeuHisArgAlaLeuHisAlaGlnGlyAsp                              305310315320                                                                  ProAlaLeuSerMetSerHisTrpMetPheHisGlnGlnAlaLeuGln                              325330335                                                                     GluTyrIleLeuMetCysCysGlnCysProAlaGlyGlyLeuLeuAsp                              340345350                                                                     LysProGlyLysSerArgAspPheTyrHisThrCysTyrCysLeuSer                              355360365                                                                     GlyLeuSerIleAlaGlnHisPheGlySerGlyAlaMetLeuHisAsp                              370375380                                                                     ValValLeuGlyValProGluAsnAlaLeuGlnProThrHisProVal                              385390395400                                                                  TyrAsnIleGlyProAspLysValIleGlnAlaThrThrTyrPheLeu                              405410415                                                                     GlnLysProValProGlyPheGluGluLeuLysAspGluThrSerAla                              420425430                                                                     GluProAlaThrAspGluGluPheMetAlaAlaThrGluGlyValGly                              435440445                                                                     GluAlaAlaGlnGlyGlyGluProGlyGlnProAlaGlnProProPro                              450455460                                                                     GlnProHisProProProProGlnGlnGlnHisLysGluGluMetAla                              465470475480                                                                  AlaGluAlaGlyGluAlaValAlaSerProMetAspAspGlyPheVal                              485490495                                                                     SerLeuAspSerProSerTyrValLeuTyrArgAspArgAlaGluTrp                              500505510                                                                     AlaAspIleAspProValProGlnAsnAspGlyProAsnProValVal                              515520525                                                                     GlnIleIleTyrSerAspLysPheArgAspValTyrAspTyrPheArg                              530535540                                                                     AlaValLeuGlnArgAspGluArgSerGluArgAlaPheLysLeuThr                              545550555560                                                                  ArgAspAlaIleGluLeuAsnAlaAlaAsnTyrThrValTrpHisPhe                              565570575                                                                     ArgArgValLeuLeuLysSerLeuGlnLysAspLeuHisGluGluMet                              580585590                                                                     AsnTyrIleThrAlaIleIleGluGluGlnProLysAsnTyrGlnVal                              595600605                                                                     TrpHisHisArgArgValLeuValGluTrpLeuArgAspProSerGln                              610615620                                                                     GluLeuGluPheIleAlaAspIleLeuAsnGlnAspAlaLysAsnTyr                              625630635640                                                                  HisAlaTrpGlnHisArgGlnTrpValIleGlnGluPheLysLeuTrp                              645650655                                                                     AspAsnGluLeuGlnTyrValAspGlnLeuLeuLysGluAspValArg                              660665670                                                                     AsnAsnSerValTrpAsnGlnArgTyrPheValIleSerAsnThrThr                              675680685                                                                     GlyTyrAsnAspArgAlaValLeuGluArgGluValGlnTyrThrLeu                              690695700                                                                     GluMetIleLysLeuValProHisAsnGluSerAlaTrpAsnTyrLeu                              705710715720                                                                  LysGlyIleLeuGlnAspArgGlyLeuSerLysTyrProAsnLeuLeu                              725730735                                                                     AsnGlnLeuLeuAspLeuGlnProSerHisSerSerProTyrLeuIle                              740745750                                                                     AlaPheLeuValAspIleTyrGluAspMetLeuGluAsnGlnCysAsp                              755760765                                                                     AsnLysGluAspIleLeuAsnLysAlaLeuGluLeuCysGluIleLeu                              770775780                                                                     AlaLysGluLysAspThrIleArgLysGluTyrTrpArgTyrIleGly                              785790795800                                                                  ArgSerLeuGlnSerLysHisSerThrGluAsnAspSerProThrAsn                              805810815                                                                     ValGlnGln                                                                     (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      MetAlaAlaGly                                                                  (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      MetAlaAlaThrGluGlyValGlyGluAlaAlaGlnGlyGly                                    1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A plasmid operably containing cDNAs encoding an αsubunit and a β subunit of a mammalian farnesyl protein transferasewhich further operably contains a DNA sequence which encodes a glutamicacid-glutamic acid-phenylalanine epitope between the cDNAs that encodethe α subunit and β subunit of the mammalian farnesylprotein transferaseenzyme, such that the glutamic acid-glutamic acid-phenylalanine epitopeis operably linked to one of the subunits upon expression of theplasmid.
 2. The plasmid according to claim 1 wherein the mammalianfarnesyl protein transferase is human farnesyl protein transferase. 3.The plasmid according to claim 2 wherein the cDNAs are contained in avector which is pRD516.
 4. A bacterium transformed with the plasmidaccording to claim 3 wherein the bacterium is an Escherichia coli and isdesignated ATCC-69091.
 5. A bacterium transformed with the plasmidaccording to claim
 2. 6. A bacterium transformed with the plasmidaccording to claim 2.